MKL-1-induced PINK1-AS overexpression contributes to the malignant progression of hepatocellular carcinoma via ALDOA-mediated glycolysis

Aldolase A (ALDOA), an important metabolic enzyme in the glycolytic pathway, plays an important role in regulating tumour metabolism. In this study, we investigated the expression pattern of ALDOA in hepatocellular carcinoma (HCC) and its biological role in tumour progression. Bioinformatics analysis, western blot (WB) and RT-qPCR were performed to detect the relative expression of ALDOA in HCC tissues and cell lines. A loss-of-function approach was used to investigate the biological function of ALDOA. The role of ALDOA on glycolysis was assessed by WB, glucose and lactate assay kits and a nude mouse xenograft model. Luciferase reporter experiment, chromatin immunoprecipitation and WB were performed to elucidate the underlying molecular. The expression level of ALODA was up-regulated in HCC tissues and cell lines. High ALDOA levels were associated with poorer patient overall survival. Mechanistic studies suggest that ALDOA is a direct target of miR-34a-5p, which can inhibit glycolysis in hepatocellular carcinoma cells by targeting the 3′UTR of ALDOA. PINK1 antisense RNA (PINK1-AS) competitively sponged miR-34a-5p to increase ALDOA expression by antagonizing miR-34a-5p-mediated ALDOA inhibition. MKL-1 acted as a transcription factor to promote the expression of PINK1-AS and ALDOA, thus promoting the deterioration of HCC cells. This study shows that high expression of ALDOA contributes to the development and poor prognosis of hepatocellular carcinoma and will be a target and potential prognostic biomarker for the treatment of HCC.

Patients and tissue samples. We collected fresh tumor tissue and paracancerous tissue from 20 HCC patients surgically resected in Hubei Cancer Hospital from January 2018 to December 2020. All tumors were classified according to the 2010 American Joint Committee on Cancer (AJCC) staging system. All patients did not receive chemotherapy or radiotherapy before surgery, and signed a written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics review committee of Hubei Cancer Hospital.
Western blotting (WB). Cells were lysed on ice with RIPA lysis buffer (Beyotime, Shanghai, China) containing PMSF, and protein concentrations were determined using the Enhanced BCA Protein Assay Kit (Meilune, Wuhan, China). Protein extracts (40 μg) were separated by electrophoresis on 12.5% SDS-polyacrylamide gels, then transferred to nitrocellulose membranes and incubated overnight with antibodies together. Secondary antibodies were added and incubated for 1 h. TBST washes were used for development. The reaction was completed using the Intelligent Chemi-TM Analysis System (Thermo Fisher Scientific, Massachusetts, USA). RT-qPCR analysis. Expression levels of ALDOA and other genes in HCC tissues and cells were measured using qRT-PCR according to the manufacturer's instructions (Yeasen, Shanghai, China). β-actin was used as a control. Primers are listed in Supplementary Table 1. Total RNA was extracted using an ultra-pure RNA kit (CWBIO, Beijing, China) and cDNA was synthesized using a HiScript II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China).
For miRNA quantification, Bulge-loop™ miRNA qRT-PCR primer sets (one RT primer and one pair of qRT-PCR primers per set) were designed by RiboBio (Guangzhou, China) specifically for miR-34a-5p. The cDNA was synthesized using the miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).
Chromatin immunoprecipitation. The chromatin immunoprecipitation (ChIP) experiment was performed using the EpiQuik ChIP kit (Epigentek) according to the manufacturer's instructions. Huh-7 and HepG2 cells were treated with formaldehyde and incubated for 10 min to produce DNA-protein crosslinks. Cell lysates are then sonicated to produce 200-300 bp chromatin fragments and immunoprecipitated using MKL-1 specific antibody or IgG as a control. The precipitated chromatin DNA is recovered and analyzed by RT-qPCR. The primers related to ChIP analysis are listed in Supplementary Table 3.
Hematoxylin-eosin staining and immunohistochemistry experiment. The tumor tissues of nude mouse were sent to Servicebio Biotech for paraffin-embedded sectioning. Hematoxylin-Eosin staining kit (Solarbio, Beijing, China) is used for hematoxylin-eosin staining (H&E). Use streptavidin alkaline phosphatase immunohistochemical staining kit (Servicebio, Wuhan, China) for immunohistochemistry (IHC) experiments. All the above operations are carried out in strict accordance with the manufacturer's instructions.
Tumor formation assay in nude mouse model. The animal experiment procedure has been approved by the Laboratory Animal Center of Wuhan University of Science and Technology and the Experimental Animal Ethics Review Committee. BALB/c-nu (nude mouse) mice obtained from Wuhan Shulb. Three-week-old nude mouse were randomly divided into two groups, each of which was injected subcutaneously with 1 × 10 8 Huh-7 cell line that stably knocks down ALDOA and the control group. After 28 days, all nude mice were euthanized by intraperitoneal injection of sodium pentobarbital and diazepam. And check the growth of subcutaneous tumors in nude mouse.
Ethics approval. The study was approved and reviewed by the Medical Ethics Committee of Wuhan University of Science and Technology and was conducted with the consent of the subjects and in accordance with the Declaration of Helsinki. All animal experiments were performed in compliance with the ARRIVE guidelines. The study was approved by the Animal Ethics Committee of Wuhan University of Science and Technology. All methods were carried out in accordance with relevant guidelines and regulations.

Statistical analysis.
All experiments in this article were repeated at 3 times. These values are expressed as mean ± standard deviation. Differences between various groups were evaluated using a two-tailed Student's t-test. Kaplan-Meier survival chart was used to draw the survival curve, and the log-rank test was used for testing. A Pearson correlation analysis was performed to analyze the correlation between ALDOA, miR-34a-5p, ALDOA and MKL-1 levels. Two-sided P values < 0.05 were considered statistically significant.

Results
Expression of ALDOA is up-regulated in human HCC tissues and correlates with poor prognosis. We assessed ALDOA transcript levels based on the Starbase database (http:// starb ase. sysu. edu. cn/ index. php) in multiple HCC studies. The results showed that mRNA levels of ALDOA were elevated in HCC tissues relative to normal tissues (Fig. 1a). Analysis of 15 HCC cohorts in the HCCDB database (http:// lifeo me. net/ datab ase/ hccdb/ home. html) also revealed that ALDOA mRNA expression was significantly higher in HCC tissues than in adjacent normal tissues (Fig. S1). The H&E staining was performed to confirm the morphological characteristics of HCC tissues and paracancerous tissues. IHC was used to detect the distribution of ALDOA in tumour and paraneoplastic tissues, and it was found that ALDOA was mainly expressed in HCC tissues and to a lesser extent in paraneoplastic tissues (Fig. 1b,c). Subsequently, ALDOA protein was measured by WB analysis and ALDOA mRNA was determined by RT-qPCR. The expression pattern of ALDOA was validated in 20 HCC patients, and ALDOA protein and mRNA showed an overall upward trend ( Fig. 1d-f). High expression of ALDOA was found to be associated with poor prognosis in HCC patients by bioinformatics analysis. The expression level of ALDOA was positively correlated with AFP expression level (Fig. 1g). In addition, there is a www.nature.com/scientificreports/ significant positive correlation between higher ALDOA levels and cancer stages (Fig. 1h). As shown in Fig. 1i, elevated ALDOA levels predicted a poorer overall survival (OS) in patients with HCC.

ALDOA is required for malignant behaviors in HCC cells.
We examined the protein and mRNA expression levels of ALDOA in the normal human hepatocyte line THLE-3 and four types of HCC cells using RT-qPCR and WB. The results showed that the expression level of ALDOA in HCC cell line was higher than that in normal cell line ( Fig. 2a-c). To assess the function of ALDOA in HCC, we identified the top 50 genes positively associated with ALDOA in HCC (Fig. S2a). The functional enrichment analysis revealed that ALDOA was significantly associated with glucose metabolic processes (Fig. S2b). We next investigated the effect of ALDOA knockdown on HCC at the cellular level. SiRNA-1 sequences targeting ALDOA showed the most pronounced knockout effect and was therefore selected for subsequent experiments ( Fig. 2d-i). We found that ALDOA silencing significantly inhibited the expression of aerobic glycolysis-related proteins ( Fig. 2j-m). We also analysed the levels of glucose and lactate in the culture medium supernatant and found that ALDOA knockdown also significantly inhibited glucose uptake and lactate production (Fig. 2n,o). This indicated that ALDOA silencing significantly inhibited aerobic glycolysis in HCC cells.  HCC growth, we designed shRNA based on the sequence of siRNA-1 targeting ALDOA to knockdown ALDOA (Fig. 3a,b). In a nude mouse xenograft model, Huh-7 cells with a lentiviral vector producing shRNA against ALDOA were inoculated subcutaneously into mouse, and Huh-7 cells infected with a lentiviral vector bearing a control shRNA were inoculated subcutaneously into another group of mouse as a control. Through this in vivo experiment, we observed that stable knockdown of ALDOA effectively inhibited tumour growth in nude mouse (Fig. 3c). This is reflected in a significant reduction in tumor size and weight (Fig. 3d,e). The expression www.nature.com/scientificreports/ of ALDOA in tumor tissues was examined by WB, which showed that ALDOA remained stably knockdown in nude mice (Fig. 3f,g). We performed histopathological analysis of tumors in nude mouse using H&E staining, and then examined the expression levels of ALDOA, PFKM and GLUT1 in nude mouse tumors by IHC. The results showed that the expression levels of ALDOA, PFKM and GLUT1 were down-regulated in sh-ALDOA group, and H&E staining showed similar change (Fig. 3h-k).

PINK1-AS targets miR-34a-5p to exert its tumor-promoting effects in HCC.
Analysis using miRcode (http:// www. mirco de. org/) website suggested that PINK1-AS could bind miR-34a-5p (Fig. 5a). We examined the expression of PINK1-AS in the 20 pairs of specimens, which was significantly up-regulated in HCC tissues (Fig. 5b). And we performed RT-qPCR to quantify the expression levels of PINK1-AS in the hepatic epithelial cell and two HCC cells. As shown in Fig. 5c, PINK1-AS was highly expressed in HCC cells, and the overall survival rate of patients with high PINK1-AS expression was poor (Fig. 5d). The expression of PINK1-AS and miR-34a-5p showed a negative correlation in tumor samples (Fig. 5e). In HCC cells, we found that knockdown the expression of PINK1-AS leads to up-regulation of miR-34a-5p (Fig. 5f,g). Inhibition of miR-34a-5p expression leads to up-regulation of PINK1-AS (Fig. 5h,i). In addition, we generated mutant sequences of PINK1-AS that were unable to bind miR-34a-5p for subsequent fluorophore enzyme reporter gene assays (Fig. 5j). As shown in Fig. 5k, miR-34a-5p mimics significantly reduced luciferase activity in HCC cells transfected with the PINK1-AS-WT sequence, whereas luciferase activity was not significantly altered in PINK1-AS-WUT transfected HCC cells. After transfection of PINK1-AS expression plasmid, the decreased luciferase activity after transfection of miR-34a-5p was restored (Fig. 5l). Bioinformatics analysis shows that the expression of PINK1-AS and ALDOA is positively correlated in tumor samples (Fig. 5m). Subsequently, the 3′UTR of ALDOA was co-transfected with the PINK1-AS plasmid and miR-34a-5p mimics. PINK1-AS overexpression negated the decrease in luciferase activity induced by overexpressing miR-34a-5p (Fig. 5n). This result implied that PINK1-AS bound to miR-34a-5p and released ALDOA from miR-34a-5p. Knockdown of PINK1-AS expression will result in downregulation of ALDOA levels (Fig. 5o,p), and inhibited the aerobic glycolysis process of tumor cells (Fig. S4a-d).

MKL-1 induces the expression of PINK1-AS and ALDOA by acting as a transcription factor.
The promoter regions of PINK1-AS and ALDOA were located and analysed through the UCSC (https:// genome. ucsc. edu/) website, the first 2000 bp upstream of the transcription start site was found to contain the MKL-1 binding site. Bioinformatics analysis shows that MKL-1 is up-regulated in HCC (Fig. 6a). The association between MKL-1 expression and survival outcomes of HCC patients was assessed using Kaplan-Meier survival curves, and analysis showed that patients with high MKL-1 expression had poor elevated overall survival (Fig. 6b). To verify whether MKL-1 mediated the expression of PINK1-AS and ALDOA, we knocked down MKL-1 using siRNA targeting MKL-1 (Fig. 6c-h). MKL-1 down-regulation resulted in a significant decrease in the expression of PINK1-AS and ALDOA in Huh-7 and HepG2 (Fig. 6i-l). Further, we found that silencing of MKL-1 significantly inhibited aerobic glycolysis in HCC cells (Fig. S5a,b). Correlation analysis showed that the expression level of MKL-1 was positively correlated with the expression levels of ALDOA and PINK1-AS in HCC tissues (Fig. 6m,n). To investigate whether MKL-1 directly transcriptionally regulates ALDOA and PINK1-AS expression, we have verified that MKL-1 can bind to the promoter regions of PINK1-AS and ALDOA by ChIP experiments (Fig. 6o,p). In addition, we constructed ALDOA and PINK1-AS promoter luciferase reporter plasmids (Fig. 6q,r). Luciferase assays showed that MKL-1 could promote PINK1-AS and ALDOA promoter luciferase activity (Fig. 6s,t).

Discussion
The reprogramming of tumour metabolism secures favourable conditions for tumour cells to survive in a new environment 22,23 . The greater glycolytic capacity in highly malignant or metastatic tumour cells suggests a potential correlation between altered tumour metabolic conditions and their ability to proliferate and migrate invasively. Increasing ALDOA expression accelerates the process of glycolysis in tumour cells, while decreasing ALDOA expression decreases ATP production and reduces cell membrane integrity 24 . In addition to regulating glycolysis and energy metabolism, ALDOA can also be involved in tumourigenesis and development through a variety of pathways. In a hypoxic environment, ALDOA can mediate EMT in tumour cells by regulating hypoxia  www.nature.com/scientificreports/ inducible factor-1α (HIF-1α), which enhances tumour cell migration 25,26 . Caspi M et al. concluded that ALDOA is a new regulator of the wnt signalling pathway that can GSK-3β-dependent mechanism to activate the classical wnt signaling pathway, which is closely related to tumorigenesis and development 27 . In this study, we used TCGA data to determine that ALDOA expression was upregulated in HCC. We then confirmed high ALDOA expression in HCC by RT-qPCR and WB analysis, high ALDOA expression in HCC tissues was associated with poor prognosis and may be an independent prognostic indicator. Through loss-of-function assays, we confirmed that ALDOA promotes glycolysis of HCC in vitro and in vivo. These results suggest that ALDOA may play an important role in the progression of HCC. Studies have confirmed that dysregulation of miRNAs plays an important role in a variety of diseases, including malignancies and inflammatory diseases 28 . In previous studies, miR-34a-5p was found to have an important role in the pathogenesis of a variety of diseases. Gao J et al. found that miR-34a-5p was lowly expressed in colon cancer patients and could target P53 to promote tumour cell metastasis and disease recurrence 29 . Jiang et al. showed that garcinia cambogia could target miR-34a-5p through up-regulation of MDM4 gene to inhibit the anti-apoptotic ability of cancer cells in non-small cell lung cancer patients, thereby eliminating or shrinking cancer foci 30 . In addition, miR-34a-5p also has a regulatory role in the pathogenesis of many benign diseases. Cosín-Tomás et al. found that miR-34a-5p was differentially expressed in the serum of patients with Alzheimer's disease and normal subjects 31 . Li et al. showed that miR-34a-5p also has an important role in diabetic nephropathy, and high expression of miR-34a-5p inhibited cell proliferation and promote renal fibrosis 32 . In this study, after bioinformatics analysis, we found that ALDOA has a binding region to miR-34a-5p in its 3′UTR, and we hypothesized that ALDOA is likely to be a target gene of miR-34a-5p. Both protein expression assays and luciferase reporter experiment verified that ALDOA is a target gene of miR-34a-5p.
Current studies have shown that lncRNAs play an important role in the regulation of gene expression, embryonic development, species evolution, material metabolism, and tumourigenesis and metastasis 16,[33][34][35] . PINK1-AS, an antisense RNA of PINK1, is regulated by PINK1-AS in the regulation of mitochondrial function. It has been reported that PINK1-AS expression is elevated in patients with multiple sclerosis 36 . Márki S et al. found that polymorphisms in the PINK1-AS gene were associated with Parkinson's disease 37 . In the present study, we found that PINK1-AS could act as a ceRNA to antagonize miR-34a-5p-mediated degradation of ALDOA and indirectly increase the expression of ALDOA mRNA. In vitro experiments also further demonstrated that PINK1-AS could promote aerobic glycolysis in HCC cells. However, under what circumstances will a lncRNAs digest or reserve the target miRNAs remain needs in-depth exploration.
MKL-1 is widely expressed in mammals and plays an important role in various physiological and pathological processes in the body 38 . Interfering with MKL-1 expression in breast cancer cells has been reported to attenuate the migration level of tumour cells 39 . The knockdown of MKL-1 in mice significantly reduced the tumorigenic ability of tumor cells and their ability to migrate to the lung with the blood circulation 40 . MKL-1 can also act as a transcription factor to promote cancer development by altering the expression of other genes in cancer cells. MKL-1 can bind directly to the core region of the CAPP1 promoter to regulate the activity of the CAPP1 promoter 41 . MKL-1 mediates TGF-β-induced RhoJ transcription to promote breast cancer cell migration and invasion 42 . In this study, we found that PINK1-AS and ALDOA are direct targets of the transcription factor MKL-1, as confirmed by the binding of MKL-1 to predicted sites in the PINK1-AS and ALDOA promoter regions and the significant induction of PINK1-AS and ALDOA promoter activity induced by MKL-1. Thus, the upregulation of PINK1-AS and ALDOA in HCC is partly attributed to MKL-1 activation during tumour progression.
In this study, we investigated the role and mechanism of ALDOA in HCC. We obtained that ALDOA is a target gene of miR-34a-5p through bioinformatic target analysis, and demonstrated that miR-34a-5p silences the expression of ALDOA by targeting its 3′UTR region, thus affecting glycolysis in HCC cells. PINK1-AS1 could act as a "sponge" for miR-34a-5p and thus inhibit the silencing effect of miR-34a-5p on ALDOA. We also found MKL-1 binding sites at the promoters of ALDOA and PINK1-AS, suggesting that MKL-1 promotes the transcription of ALDOA and PINK1-AS by targeting their promoters. In HCC cells, PINK1-AS and ALDOA levels are consistently parallel, with increased levels promoting aerobic glycolysis.

Conclusion
In conclusion, we comprehensively investigated the functional role and molecular mechanism of ALDOA in HCC. Our results suggest that ALDOA is up-regulated in HCC cell lines and tissues. High ALDOA expression levels are an independent prognostic factor for the overall survival of HCC patients. Our study shows that MKL-1/PINK1-AS/miR-34a-5p/ALDOA form a circuit in cells to co-regulate glycolysis in HCC cells. Our findings support the idea that ALDOA plays a key role in HCC progression and is a potentially effective target for the treatment of HCC.

Data availability
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.